Project Summary The work proposed in this application will seek to develop a novel and greatly improved bioreactor to study events in the upper intestine. Specifically, we will validate the model by studying the effect of IgA on S. typhimurium attachment and colonization of the small intestinal villi. S. typhimurium is an enteric pathogen that kills as many as 400 people annually in the United States and makes upwards of a million people sick. There is a great deal of knowledge available regarding S. typhimurium attachment to the upper intestine with respect to the biochemical landscape of the intestine, but there is a gap in understanding as to how IgA works in concert with toll-like receptors to prevent S. typhimurium attachment. This work will make use of novel 3D printed bioreactors that have features of well-differentiated human intestinal organoids (HIOs) and cadaveric human enteroids (HEs) growing on villous scaffolds of the appropriate size and distribution to represent actual villi, apical and basolateral flow and real time measurements of trans-epithelial electric resistance (TEER). Importantly, these reactors can be used to add complex fluidic properties of the small intestine into studies around S. typhimurium attachment to the upper intestinal epithelial cells in a well-controlled and reproducible manner. The overall objective for this research is to culture monolayers of HIOs and HEs on villous scaffolds in a 3D printed fluidic model to better understand how fluid dynamics and epithelial gene expression contribute to bacterial niche establishment. The work will investigate S. typhimurium attachment and colonization in the presence and absence of isotopes of immunoglobulin A (IgA). The specific aims for this work are: Specific Aim 1: Create permissive growth environment for HIOs and HEs in bioreactors and validate effects of flow on growth and differentiation. Specific Aim 2: Test if the bioreactor set up can be used to study the protective effects of Sal-4 IgA against ST infection. The overarching hypothesis for this work is that combining human organoid and enteroid cells with the correct villous topography in a non-laminar flow field will lead to a far superior experimental setup for understanding the complex interplay between upper intestinal bacteria, epithelial cells and the immune system. This high risk work, if successful, will lead to a high throughput platform that will facilitate much deeper understanding of the mechanisms at play in intestinal homeostasis.